To exploit the full potential of human pluripotent stem (hPS) cells for regenerative medicine, developmental biology, and drug discovery, defined conditions for hPS cell propagation and differentiation must be reproducible and therefore chemically defined. To this end, the chemical cues that instruct hPS cells to make specific decisions must be determined. While strategies to identify the soluble cues are well established, an understanding of how insoluble signals guide cell fate decisions is lacking. Chemical synthesis provides access to well-defined surfaces and materials that vary in binding epitope identity, density, and in physicochemical properties. The three Specific Aims have been devised to leverage synthetic methods from chemistry and chemical biology to generate tailored materials and surfaces that promote hPS cell expansion or differentiation to desired cell types. In the past grant period, chemically defined peptide- substituted surfaces that promote hPS cell propagation were identified. The most effective substrates interact with hPS cell surface glycosaminoglycans. The experiments proposed in Aim 1 were conceived to test the hypothesis that engagement of hPS glycosaminoglycans contributes to pluripotency. In Aim 2, we shall employ our surface array screen to identify insoluble signals (peptide- or small molecule-derived) that act in concert with soluble signals to direct hPS cell differentiation. In Aim 3, we shall synthesize a series of biocompatible polymers that vary in cell binding groups, pore size, and elasticity to address how physicochemical cues influence cell fate decisions. A novel feature of the approach is the use of an array strategy to identify new surfaces that act on hPS cells to direct their proliferation and differentiation. The proposed experiments leverage this surface array screen not only to identify functional surfaces but also to devise and test novel hypotheses regarding the signaling pathways and molecular mechanisms that underlie hPS self-renewal and differentiation. Progress in understanding how hPS cells respond to distinct combinations of soluble, insoluble, and mechanical cues will be transformative: It will advance applications of hPS cells to human disease.